Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2021 Feb 5;49(4):1935–1950. doi: 10.1093/nar/gkab030

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

PMC Copyright notice

Figure 5. — PAUPAR directly binds to genomic region of target neural genes. (A) Percent recovery of PAUPAR following ChIRP on day 16 of dorsal neural differentiation. GAPDH served as negative control. (B) ChIRP assay using probes targeting PAUPAR or LacZ mRNA followed by QRT-PCR on SOX1 genome (G) (TSS +1300), SOX1 distal region (D) (TSS -4000), POU3F3 G (TSS +1000), POU3F3 D (TSS -4000), MSX1 G (TSS +1000), MSX1 D (TSS –4000), NR2F1 G (TSS +1500) and NR2F1 D (TSS –4000) genomic region on day 16 (left); TBR1 G (TSS +200), TBR1 D (TSS –4000), TBR2 G (TSS -1000) and TBR2 D (TSS -4000) genomic region on day 25 (right) of dorsal neural differentiation. GAPDH (TSS +3500) served as negative control. (C) In vitro triplex pulldown assay showed the binding between PAUPAR and SOX1 piece1–6 fragments (Piece1 -416/+90, Piece2 +60/+470, Piece3 +431/+1023, Piece4 +1074/+1713, Piece5 +1721/+2289, Piece6 +2305/+2760). GAPDH fragment (+3094/+3583) served as negative control. (D) Schematic representation of the deletion mutants of PAUPAR. (E) In vitro triplex pulldown assay showed the binding between SOX1 piece4 fragment and full-length PAUPAR or PAUPAR deletion mutants (A, B, C, D, or E). (F) In vitro triplex pulldown assay showed the binding between SOX1 piece4 fragment and full-length PAUPAR or PAUPAR 5′ region deletion mutant. (G) MS2bp-YFP RNA pull-down analysis showed the binding between Flag-PAX6a and PAUPAR 5′ region deletion mutant in 293FT extracts. (H) QRT-PCR analysis showed the overexpression of PAUPAR deletion mutant in the PAU-PAKI cells. (I) Expression of SOX1 after PAUPAR or PAUPAR deletion mutant overexpression in the PAU-PAKI cells on day 16 of dorsal neural differentiation. (J) Enrichment of PAX6 on SOX1 (TSS +1300) genomic region after PAUPAR or PAUPAR deletion mutant overexpression in the PAU-PAKI cells on day 16 of dorsal neural differentiation. (K, L) Immunostaining assay of PAX6 (green) and SOX1 (red) after PAUPAR or PAUPAR deletion mutant overexpression in the PAU-PAKI cells on day 16 of dorsal neural differentiation (K), and quantification of PAX6⁺SOX1⁺ cells (L). Scale bar, 100 μm. Data are presented as mean ± SEM. *P< 0.05, **P< 0.01, ***P< 0.001 versus GAPDH group (B), or versus negative group (C), or versus PAUPAR group (E and F), or versus CTRL group (I, J and L); ^##P< 0.01, ^###P< 0.001 versus PAU-PAKI + ptPAU-dox group (I, J and L) (t-test/ANOVA). See also Supplementary Figure S5.